EA012573B1 - Rnamodulation of rsv and therapeutic uses thereof - Google Patents

Abstract

The present invention is based on the in vivo demonstration that RSV can be inhibited through intranasal administration of iRNA agents as well as by parenteral administration of such agents. Further, it is shown that effective viral reduction can be achieved with more than one virus being treated concurrently. Based on these findings, the present invention provides general and specific compositions and methods that are useful in reducing RSV mRNA levels, RSV protein levels and viral titers in a subject, e.g., a mammal, such as a human. These findings can be applied to other respiratory viruses.

Description

The application for this invention claims the priority advantage of provisional application US No. 60/642364, filed January 7, 2005, which is incorporated herein by reference in full.

Technical field

The invention relates to the field of treatment of respiratory syncytial viruses (Β8ν) and to compositions and methods for modulating virus replication, and more particularly, to negative regulation of the gene (s) of the respiratory syncytial virus by oligonucleotides by RNA interference, introduced locally into the lungs and nasal passage by inhalation / intranasal or by injection / intravenously.

Background

Due to the natural function performed, the airways are exposed to many pathogens in the air that cause various respiratory diseases. Respiratory tract viral infection is the most common cause of hospitalization of young children in developed countries with approximately 91,000 annual hospitalizations in the United States worth $ 300 million. Human respiratory syncytial virus (K.8U) and parainfluenza virus (Ρίν) are the two main factors for respiratory diseases; together they infect the upper and lower respiratory tract, leading to croup, pneumonia and bronchiolitis (Oreizyet, Ρ.Ι.Μ. Vekr1g. Vek. 3 (8irr1 1), 315-320 (2002), EakUi, A.Ts! ., Οΐίη. ΜίετοΜοΙ. Veu. 17, 390-412 (2004)). Only Β8ν infects up to 65% of all children in the first year of life and virtually all children in the first 2 years. Β8ν is an important cause of morbidity and mortality in older people as well. Immunity after Β8ν infection is neither complete nor lasting, and therefore, repeated infections occur in all age groups. Children who have had ν8ν with bronchiolitis are more likely to develop wheezing and asthma at a later age. The search for an effective treatment and vaccine against Β8ν lasted for almost four decades and had little success (Orepkyayet, Ρ.Ι.Μ. Vekr1g. Vek. 3 (8irr1 1), 815-820 (2002), Maddop, K. e! A1 ., Βеν. Furniture. Νίτοί. 14, 149-168 (2004)). No vaccines against Β8ν have been approved at this time. Strains of both viruses also exist in non-human animals, such as cattle, goats, pigs and sheep, which leads to losses in agriculture, dairy and meat industry (Eak! Op, A.1., E! A1 ., Οΐίη. ΜίετοΜοΙ. Ge. 17, 390-412 (2004)).

Both Β8ν contain negative segmented non-segmented RNA genomes and belong to the paramyxovirus family. A number of features of these viruses contribute to the difficulty of prevention and treatment. Viral genomes mutate at a high speed due to the lack of a mechanism for adjusting the reading of RNA genomes, representing significant difficulty in creating a reliable vaccine or antiviral drug (8 and 11beg, XV.Μ. Οΐίη. Μίετοϋίοΐ. Βον. 13, 1-15 (2000)). Promising inhibitors of the fusion protein (E) Β8ν were partially abandoned because the virus developed stable mutations that were mapped to the E gene (ΒαζίηΕον. Ν., Е !. a1., Αηΐίνίτ. Century 55, 189-200 (2002), Μοήοη, C. 1. E! A1. Νίτο1οβν 311, 275-288 (2003)). Both viruses are associated with cellular proteins, which makes it difficult to obtain cell-free viral material for vaccination (Wigke, E., e! A1., Νίτο1ο ^ 252, 137-148 (1998), Wigke, E., e! A1., 1 U1go1. 74, 669-675 (2000), Sir1a, 8., e! A1., 1. U1to1. 72, 2655-2662 (1998)). Finally, the immunology of both, and especially the immunology of Β8ν, is extremely complex (ReeЫek, Β.8., 1g., E! A1., Uia1. Tyutite 16, 25-34 (2003), Science Century, Ε.Μ., e ! a1., 1. νίτο1. 11, 98319844 (2003)). The use of Β8ν proteins as vaccines leads to “immunopotentiation” or to a vaccine-enhanced disease (ΡοΗε ^ EP! A1. 1. Exp. 6ο.6. 196, 859-865 (2002)). In general, the problem is underestimated due to the recent closure of many biopharmaceutical programs against Β8ν.

The Β8ν genome contains a single reverse strand of RNA with a length of 15222 nucleotides producing eleven basic proteins (Ea1keu, Α.Β. : 371-84.) Two of these proteins, glycoproteins E (fused) and C (binding), are the main surface proteins and the most important for the induction of protective immunity. 8H (small hydrophobic) protein, Μ (matrix) protein, and M2 (22 kDa) protein are bound to the envelope of the virus, but do not induce a protective immune response. Proteins N (the main protein bound to the nucleocapsid), Ρ (phosphoprotein), and b (the main polymerase protein) were found to be associated with virion RNA. Two non-structural proteins N81 and N82, apparently, are involved in the interaction of the host and the virus, but are absent in the infectious virions.

Human strains Β8ν were divided into two main groups: A and B. It was shown that glycoprotein C is the most divergent among Β8ν proteins. It is believed that the variability of glycoprotein C Β8ν between two groups of Β8ν and within these groups plays an important role in the ability of Β8ν to cause annual outbreaks of the epidemic. Glycoprotein C contains 289-299 amino acids (depending on strain Β8ν) and has an intracellular, transmembrane and highly glycosylated stalk-like structure of 90 kDa, as well as heparin binding domains. This glycoprotein exists in secreted and membrane bound forms.

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Currently, there are no effective methods for treating P8U infection (Maddop K. Aiy Vapk 8., 2004, Sun \ tc \\ b ъη Meyua1 Ulgoodu. 14: 149-68). Infection of the lower respiratory tract K.8U in most cases can be cured independently. There are no clear guidelines or criteria for the treatment or hospitalization and discharge of young and older children with this disease. Hypoxia. which may occur in connection with K.8U infection. can be treated with oxygen through a nasal catheter. Mechanical ventilation in children with respiratory failure. shock or recurrent apnea can reduce mortality. Some physicians prescribe steroids. However, some studies have shown. that steroid treatment does not affect the course of the disease in young and old children. hospitalized with bronchiolitis. Consequently. corticosteroids alone or in combination with bronchodilators can be useless in the treatment of bronchiolitis in otherwise healthy patients. not receiving mechanical ventilation. Steroids have also been used in young and older children with primary cardiopulmonary diseases. such as bronchopulmonary dysphasia and asthma.

Ribavirin guanosine analog. possessing antiviral activity. used to treat young and older children with P8U bronchiolitis since the mid-1980s .. but many studies. evaluating its use. showed conflicting results. In most centers, the use of ribavirin is currently limited to immunocompromised and critically ill patients.

The severity of P8U bronchiolitis was associated with low serum concentrations of retinol. but studies in hospitalized children with P8U bronchiolitis have shown. that the addition of vitamin A does not provide a positive effect. Therapeutic studies of 1500 mg / kg of intravenously administered P8U immunoglobulin or 100 mg / kg of inhaled immunoglobulin with P8U infection of the lower respiratory tract also did not show significant beneficial effects.

In developed countries, treatment of P8U lower respiratory tract infection is generally limited to symptomatic therapy. Antiviral treatment is usually limited to life-threatening situations due to the high cost and lack of consensus on effectiveness. In developing countries, oxygen is the main treatment (when available) and prevention is the only way to reduce mortality.

RNA interference or “RNA1” is a term. originally proposed by P1ge and co-workers to describe scientific observations. that double-stranded RNA (d & RNA) can block gene expression when injected with worms (P1ge e! a1 .. IA1ige 391: 806-811. 1998). Short jcRNA directs gene-specific post-transcriptional silencing in many organisms. including in vertebrates. and introduces a new tool for studying gene function. RNA1 has been proposed as a way to develop a new class of therapeutic agents. However, to date, this has largely remained an assumption without demonstrating evidence of the potential therapeutic use of RNA1.

Thus. There is a need for safe and effective vaccines against P8U. especially for younger and older children. There is also a need for drugs and methods of treating P8U infection in all age groups and in individuals with immunodeficiency. There is also a need for scientific methods to characterize a protective immune response to P8U to study the pathogenesis of this disease and to facilitate screening of drugs and vaccines. The present invention overcomes the disadvantages. prior art. providing methods and compositions. effective for modulating or preventing P8U infection. In particular. the present invention contributes to the prior art. offering 1RNA agents. which. as shown. reduce the levels of P8U ίη νίίτο and ίη νίνο. and are also effective against the two main subtypes of P8U and demonstrate the therapeutic activity of molecules of this class.

SUMMARY OF THE INVENTION

The present invention is based on ίη νίίτο and ίη νίνο demonstrating that. that P8U can be inhibited by intranasal administration of 1RNA agents. as well as parenteral administration of such agents. and identification of effective 1RNA agents from P. N and L of the P8U gene. which can lower RNA levels. both with subtype A. and with subtype B P8U. Based on the results. The present invention relates to particular compositions and methods. applicable to reduce P8U mRNA levels. P8U protein levels and P8U virus titers in a patient. for example a mammal. such as a person.

The present invention particularly relates to 1RNA agents. mainly consisting of or containing at least 15 or more adjacent nucleotides of one of the P8U genes. especially the genes P. N and L P8U. and more specifically. to agents. containing 15 or more adjacent nucleotides of one of the sequences. presented in table. 1 (a-s). The 1RNA agent preferably consists of less than 30 nucleotides per strand. for example 21-23 nucleotides. such as indicated in table. 1 (a-s). A double-stranded 1RNA agent may have blunt ends, or more preferably have protrusions of 1-4 nucleotides from one or both 3'-ends of this agent.

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In addition, the 1RNA agent can either contain only natural ribonucleotide subunits, or can be synthesized in such a way that it contains one or more modifications of the sugar or base of one or more ribonucleotide subunits included in this agent. The 1RNA agent can additionally be modified so that it binds to a ligand that is selected to improve the stability, distribution, or incorporation of this agent into the cell, for example, cholesterol. 1RNA agents may additionally be in isolated form or may be part of a pharmaceutical composition used for the methods described herein, in particular as a pharmaceutical composition made for delivery to the lungs or nasal passage or made for parenteral administration. The pharmaceutical compositions may contain one or more 1RNA agents and, in some embodiments, will contain two or more 1RNA agents, each of which is directed to different segments of the P8U gene or to two different P8U genes.

The present invention further relates to methods for reducing K.8U viral mRNA levels in a cell. Such methods include the step of administering to the patient one of the 1RNA agents of the present invention, as hereinafter further described below. The methods of the present invention utilize cellular mechanisms involved in RNA interference to selectively destroy viral mRNA in a cell and include the step of contacting the cell with one of the antiviral 1RNA agents of the present invention. Such methods can be performed directly on the cell, or they can be performed on a mammalian patient by administering to the patient one of the 1RNA agents / pharmaceutical compositions of the present invention. The decrease in viral mRNA in cells results in a decrease in the amount of viral protein produced and in the body leads to a decrease in the titer of the replicating virus (as shown in the examples).

The methods and compositions of the invention, for example the methods and compositions of the 1RNA agent, can be used at any dose and / or composition described herein, as well as any route of administration described herein. Of particular importance is the intranasal administration of the 1RNA agent shown here and its ability to inhibit virus replication in respiratory tissues.

A detailed description of one or more embodiments of this invention is given in the accompanying drawings and in the description below. Other features, objects, and advantages of this invention will be apparent from the description, drawings, and claims. The present invention includes all cited references, patents and patent applications as references in full for any purpose.

Brief Description of the Drawings

FIG. 1. Ιη νίΐτο inhibition of P8U using 1RNA agents. 1RNA agents are presented in table. 1 (a-c) were tested for anti-CBU activity in the plaque assay described in the examples. Each column (column) represents 1RNA agent, are given in table. 1 (ac), for example, column 1 is the first agent in table. 1a, etc. Active 1RNA agents have been identified.

FIG. 2. Dose-dependent inhibition of P8U ίη νίΐτο using 1RNA agents. Tested examples of active agents from the table. 1 on anti-CBU activity in the study of plaque formation, in four concentrations, as described in the examples. A dose-dependent response was found with the tested active 1RNA agents.

FIG. 3. Ιη νίΐτο inhibition of the P8U subtype B using 1RNA agents. 1RNA agents of FIG. 2, were tested for anti-CBU activity against subtype B in the plaque assay described in the examples. Test 1RNA agents inhibited subtype B.

FIG. 4. Ιη νίνο inhibition of P8U using 1RNA agents. Agents described in this figure. were tested for anti-CBU activity in a mouse model, as described in the examples. 1RNAs effectively reduced the titers of the virus ίη νίνο.

FIG. 5. Ιη νίνο inhibition of K8U using L-OR-1730. L-OR-1730 was tested in relation to the activity depending on the dose using the methods described in the examples. These agents showed a dose-dependent response.

FIG. 6. Ιη νίνο inhibition of P8U using 1RNA agents. The 1RNA agents described in this figure were tested for anti-CBU activity of ίη νίνο, as described in the examples.

FIG. 7. Ιη νίνο inhibition of P8U using 1RNA agents. The 1RNA agents described in this figure were tested for anti-CBU activity of ίη νίνο, as described in the examples.

FIG. 8A. Ιη νίνο inhibition of P8U using 1RNA agents with local administration.

FIG. 8B. Ιη νίνο inhibition of P8U using 1RNA agents when delivered by aerosol. The 1RNA agents described in this figure were tested for anti-CBU activity of ίη νίνο, as described in this example.

FIG. 9. Ιη νίνο protection against P8U infection using 1RNA agents. The 1RNA agents described in this figure were tested before infection with P8U to study the protective effect.

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Detailed description

For convenience of presentation, the term "nucleotide" or "ribonucleotide" is sometimes used here with respect to one or more subunits of an RNA agent. It will be understood that the term “ribonucleotide” or “nucleotide” here may, in the case of a modified RNA or nucleotide substitute, also refer to a modified nucleotide or substitution part, further described below, in one or more positions.

The “RNA agent” as used herein is unmodified RNA, modified RNA, or a nucleoside substitute, each of which is described herein or is well known in the field of RNA synthesis. Although numerous modified RNAs and substitute nucleosides have been described, preferred examples include those that are more resistant to degradation by nucleases than unmodified RNAs.

Preferred examples include those that have a 2'-modification of sugar, a modification of the protrusions of a single chain, preferably 3 'of the protrusions of a single chain, or, in particular, in the case of a single-chain 5'-modification, which includes one or more phosphate groups or one or more analogs of a phosphate group.

As used herein, the term “1RNA agent” (short for “interfering RNA agent”) is an RNA agent that can negatively regulate the expression of a target gene, for example, CBU. Without being bound by theory, a 1RNA agent can act through one or a number of mechanisms, including post-translational cleavage of the target mRNA, sometimes referred to in this area as RNA1, or through pre-transcriptional or pre-translational mechanisms. The 1RNA agent may be a double-stranded άδ 1RNA agent.

The “άδ 1RNA agent” (abbreviated from “double-stranded 1RNA agent”) as used herein is a 1RNA agent that includes more than one and preferably two helices in which hybridization within a strand can form a portion of a duplex structure. “Spiral” herein refers to a continuous sequence of nucleotides (including non-natural or modified nucleotides). Two or more chains can be separate molecules, or each form a part, or they can be covalently linked, for example, by a linker, for example polyethylene glycol, to form one molecule. At least one strand may include a region that is sufficiently complementary to the target RNA. Such a chain is called an “antisense spiral”. The second strand enclosed in the b5RNA agent that contains the complementarity site for the antisense strand is called the “sense strand”.

However, the άδ 1 RNA agent can also be formed from a single RNA molecule, which is at least partially self-complementary, forming, for example, a hairpin or long narrow protruding structure including a duplex region. In this case, the term “helix” refers to one of the regions of an RNA molecule that is complementary to another region of the same RNA molecule.

Although long άδ 1RNA agents in mammalian cells can cause an interferon response, which is often damaging, short άδ 1RNA agents do not trigger an interferon response, at least not to the extent that the cell and / or host organism is harmed. 1RNA agents of the present invention include molecules that are short enough to not trigger a damaging interferon response in mammalian cells. Thus, the introduction of a 1RNA agent composition (eg, formulated as described herein) into mammalian cells can be used to suppress CBU gene expression, avoiding the damaging interferon response. Molecules that are short enough that they do not trigger a damaging interferon response are referred to herein as δ ^ RNA agents or δ ^ RNA. As used herein, <“1RNA agent” or <“1RNA” refers to a 1RNA agent, for example, άδ 1RNA agent, which is short enough that does not cause a damaging interferon response in human cells, for example, has a double-stranded region of less than 30 nucleotide pairs.

The isolated 1RNA agents considered here, including άδ 1RNA agents and δ ^ RNA agents, can mediate gene suppression, for example, by destroying RNA. For convenience, such RNA is also referred to herein as suppressed RNA. Such a gene is also called a target gene. Preferably, the suppressed RNA is a CBU gene product, in particular a P, N or b gene product.

As used herein, the phrase “mediates RNA1” refers to the ability of an agent to specifically inhibit a target gene sequence. "Suppression of the target gene" means the process by which a cell, without contacting with this agent, contains and / or secretes a specific product of the target, in contact with this agent, will contain and / or secrete at least 10, 20, 30, 40 , 50, 60, 70, 80, or 90% less of such a gene product than a similar cell that has not been in contact with this agent. Such a product of the target gene, for example, may be messenger RNA (mRNA), a protein, or a regulatory element.

In antiviral applications of the present invention, the suppression of the target gene will lead to a decrease in the "titer of the virus" in the cell or in the patient. As used herein, “reduction in virus titer” refers to a decrease in the number of viable viruses produced by a cell or found in an organism in which a viral target gene has been suppressed. A decrease in the cellular amount of the virus produced will preferably result in a decrease in the measurable amount of virus produced in the tissues of the patient being treated and a decrease in the severity of the symptoms of the viral infection. 1RNA agents of the present invention are also called "antiviral (RNA agents").

As used herein, the “K.8U gene" refers to any gene identified in the genome of the B8U virus (see Eakeu, L.K., aib \ Ua1b11 E.E., 2000, Syssa1 M | sgoyuoshchota1 Isu1C \\ b. 13: 371- 84). These genes are well known in the art and include the Ν, P and b genes, which are shown here as examples.

As used herein, the term “complementarity” is used to mean a sufficient degree of complementarity for stable and specific binding between a compound of the invention and a target RNA molecule, for example, a B8U virus mRNA molecule. For specific binding, a sufficient degree of complementarity is required to avoid nonspecific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of ίη νίνο studies or therapeutic effects or in the case of ίη νίίτο studies, in the conditions under which these studies are conducted. Non-target sequences usually differ by at least 4 nucleotides.

The 1RNA agent used here is “sufficiently complementary” to a target RNA, for example a target mRNA (for example, a B8U mRNA target), if the 1RNA agent reduces the production of the protein encoded in the cell by the target RNA. The 1RNA agent can also be “completely complementary” to the target RNA, for example, this target RNA and 1RNA agent hybridize preferably to form a hybrid formed exclusively from Watson-Crick base pairs in the region of complete complementarity. A "sufficiently complementary" 1RNA agent may include an internal region (for example, at least 10 nucleotides) that is fully complementary to the viral target RNA. In addition, in some embodiments, the 1RNA agent specifically recognizes a single nucleotide difference. In this case, this 1RNA agent only mediates RNA1 if complete complementarity is found in the region (for example, within 7 nucleotides) with a difference of one nucleotide. Preferred 1RNA agents will be based on the sense and antisense sequences presented in the examples, or consist or contain them.

As used herein, the term “substantially identical” when used with respect to the first nucleotide sequence compared to the second nucleotide sequence means that the first nucleotide sequence is identical to the second nucleotide sequence, with the exception of not more than one, two or three nucleotide substitutions (for example, adenosine is substituted by uracil )

As used herein, the term “patient” refers to a mammal that is being treated for a disorder mediated by viral expression, such as a B8U infection, or that has been given prophylactic treatment to prevent a viral infection. The patient may be any mammal, such as a primate, cow, horse, mouse, rat, dog, pig, goat. In a preferred embodiment, the patient is a human.

The treatment of V8U infection used here refers to the improvement of any biological or pathological indicators, 1) which are partially mediated by the presence of the virus in the patient’s body, and 2) the outcome of which may be affected by a decrease in the level of the present virus gene products.

Creation and selection of 1RNA agents.

The present invention is based on the demonstration of the suppression of the target gene of the respiratory virus ίη νίνο after local injection of the 1RNA agent into the lungs and nasal passages either by intranasal administration / inhalation or systemically / parenterally by injection and, as a result, the treatment of viral infection. The present invention further extends to the use of 1RNA agents for several respiratory viruses and the treatment of both viral infections by co-administration of two or more 1RNA agents.

Based on these results, this invention particularly relates to a 1RNA agent that can be used in the treatment of viral infection, in particular respiratory viruses and in particular K5U infection, in isolated form and in the form of the pharmaceutical composition described below. Such agents will include a sense strand having at least 15 or more adjacent nucleotides complementary to the viral gene, and an antisense strand having at least 15 or more adjacent nucleotides complementary to the sense strand sequence. Especially effective are 1RNA agents, essentially consisting of the nucleotide sequence of the genes P N and L B8U, are given in table. 1 (ac), or containing it.

1RNA agents of the present invention are based and contain at least 15 or more adjacent nucleotides from one of the 1RNA agents, the activity of which is shown in table. 1 (a-s). In such agents, this agent may consist essentially of the complete sequence shown in this table, or contain it, or may contain 15 or more adjacent residues shown in table. 1 (ac) along with additional nucleotides of adjacent sections of the target gene.

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1RNA agent can be rationally created on the basis of sequence information and the desired characteristics and information presented in table. 1 (a-s). For example, a 1RNA agent can be created in accordance with the sequence of agents shown in the tables, as well as taking into account the complete coding sequence of the target gene.

Accordingly, the present invention relates to 1RNA agents comprising a sense strand and an antisense strand, each containing a sequence of at least 15, 16, 17, 18, 19, 20, 21 or 23 nucleotides, which is essentially identical as as defined above, portions of the respiratory virus gene, in particular the P, N or L genes of the B8U protein. Typical 1RNA agents include agents containing 15 or more adjacent nucleotides of one of the agents shown in table. 1 (a-s).

The antisense strand of the 1RNA agent must be equal in length or at least 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. Its length should be equal to or less than 50, 40, or 30 nucleotides. Preferred ranges are 15-30, 17-25, 19-23 and 19-21 nucleotides in length. Typical 1RNA agents include a chain containing 15 or more nucleotides of one of the antisense chains of one of the agents shown in table. 1 (a-s).

The sense strand of the 1RNA agent must be equal to or at least 15, 16 17, 18, 19, 25, 29, 40, or 50 nucleotides in length. Its length should be equal to or should be less than 50, 40, or 30 nucleotides. Preferred length ranges are 15-30, 17-25, 19-23 and 19-21 nucleotides. Representative 1RNA agents include strands containing 15 or more nucleotides from one of the sense strands of one of the agents shown in Table 1. 1 (a-s).

The double-stranded region of the 1RNA agent must be equal to or at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 50 nucleotide pairs long. Its length should be equal to or less than 50, 40 or 30 pairs of nucleotides. Preferred length ranges are 15-30, 17-25, 19 to 23 and 19-21 nucleotides.

The length of each chain of agents shown in the table. 1 (ac) is 21 nucleotides. 1RNA agents contain a double-stranded region of 19 nucleotides with a 2-nucleotide protrusion at each 3'-end of the agent. These agents can be modified, as described herein, to obtain equivalent agents containing at least a portion of these sequences (15 or more adjacent nucleotides) and / or modifications with respect to nucleotide bases and bonds.

Basically, the 1RNA agents of the present invention include a region of sufficient complementarity to the virus gene, for example P, N or L protein P8U. whose nucleotide length is sufficient for this 1RNA agent or its fragment to mediate the downregulation of a specific viral gene. The antisense strands of the 1RNA agents of the present invention are preferably completely complementary to the mRNA sequences of the virus gene, as is the case for the P, b or N protein of Βδν. However, the ideal complementarity between the 1RNA agent and the target is not necessary, but the correspondence should be sufficient to enable the 1RNA agent or its cleavage product to carry out direct specific sequence silencing, for example, by means of RNA1 Κ5ν mRNA cleavage.

Therefore, the 1RNA agents of the present invention include a sense strand and an antisense strand, each containing a sequence of at least 16, 17 or 18 nucleotides, which are essentially identical, as defined below, to one of the virus gene sequences, in particular P , N or L protein Βδν, such as the agent shown in table. 1 (a-c), except that no more than 1, 2, or 3 nucleotides in the chain are respectively replaced by other nucleotides (e.g., adenosine is replaced by uracil), essentially retaining the ability to inhibit K8U expression in cultured human cells, as defined below. These agents, therefore, will contain at least 15 or more nucleotides that are identical to one of the virus gene sequences, in particular the P, b or N Βδν protein genes, but 1, 2 or 3 paired bases have been introduced contrary to the principle of complementarity with respect to either virus mRNA targets, or between the sense and antisense chains. Non-complementary pairings with the target sequence of the mRNA of the virus, especially in the antisense strand, are most permissible in the terminal regions and, if present, are preferably located in the terminal region or regions, for example, within 6, 5, 4 or 3 nucleotides 5'- and / or 3 the 'end, most preferably within 6, 5, 4 or 3 nucleotides of the 5'-end of the sense strand or the 3'-end of the antisense strand. The sense chain should only be sufficiently complementary to the antisense chain to preserve the double-helical nature of the molecule along its entire length.

Preferably, the sense and antisense strands are chosen so that the 1RNA agent includes one strand or unpaired region at one or both ends of this molecule, for example, given as an example in table. 1 (a-s). Therefore, the 1RNA agent contains sense and antisense strands, preferably paired in such a way that they contain a protrusion, for example, one, or two 5 'or 3' protrusions, but preferably a 3 'protrusion of 2-3 nucleotides. Most embodiments will have a 3 'protrusion. Preferred 51 RNA agents will have single stranded protrusions, preferably 3 'protrusions from 1 to 4, or preferably 2 or 3 nucleotides in length, per

- 6 012573 one or both ends of the 1RNA agent. The protrusions may be the result of one chain being longer than the other, or the result of two chains of the same length being offset. The 5'-ends are preferably phosphorylated.

The preferred length of the duplex portion is from 15 to 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length, for example, in the series of DRR agents discussed above. Also included are embodiments in which two strands of the DRRNA agent are linked, for example covalently linked. A hairpin or other single-stranded structures representing the desired double-stranded region and preferably a 3 'protrusion are also included in this invention.

Assessment of 1RNA candidate agents.

The ability of a candidate candidate 1RNA to downregulate gene expression can be assessed. For example, a candidate 1RNA agent can be provided and reacted with a cell, for example, a human cell that has been or will be infected with a virus of interest, for example, a virus containing a target gene. Alternatively, the cell can be transfected with a construct expressing the viral target gene, thereby avoiding the need for viral infection of the model. The expression level of the target gene before and after contact with the candidate 1RNA agent can be compared, for example, by RNA, protein level or virus titer. If it is determined that the amount of RNA, protein or virus expressed from the target gene is less after contact with the 1RNA agent, then it can be concluded that this 1RNA agent negatively regulates the expression of the target gene. The level of the viral target RNA or viral protein in the cell or virus titer in the cell or tissue can be determined by any desired method. For example, target RNA levels can be determined by Northern blotting, reverse transcription polymerase chain reaction (RT-RCR), LDNA analysis or RNase protection analysis. Protein levels can be determined, for example, by Western blot analysis or by immunofluorescence. The virus titer can be determined in the analysis of plaque formation.

Stability testing, modification and retesting of 1RNA agents.

The candidate 1RNA agent can be evaluated for stability, for example, its susceptibility to cleavage with an endonuclease or exonuclease, for example, when a 1RNA agent is administered to a patient. Methods can be used to identify sites subject to modification, in particular cleavage, for example cleavage by the action of a component within the patient.

After identification of sites susceptible to cleavage, a new 1RNA agent can be constructed and / or synthesized in which a possible cleavage site is made resistant to cleavage, for example, by introducing a 2'-modification at the cleavage site, for example, a 2'-O-methyl group . This new 1RNA agent can be retested for stability and this process can be repeated until it is discovered that RNA1 exhibits the desired stability.

Testing ίη νίνο.

The 1RNA agent identified as capable of inhibiting viral gene expression can be tested for the functional activity of ίη νίνο in an animal model (e.g., a mammal, e.g., a mouse, rat, or primate), as shown in the examples. For example, a 1RNA agent can be administered to an animal, and a 1RNA agent can be evaluated for its biodistribution, stability, and its ability to inhibit the expression of a virus gene, for example, K.8U or to reduce the titer of a virus.

The 1RNA agent can be introduced directly into the target tissue, for example, by injection, or the 1RNA agent can be introduced into the animal model in the same way that it would be administered to humans. As shown here, this agent can preferably be administered by inhalation as an agent for treating a viral infection.

The 1RNA agent can also be evaluated with respect to its intracellular distribution. Such an assessment may include determining whether a 1RNA agent has entered the cell. Such an assessment may also include determining the stability (e.g., half-life) of the 1RNA agent. Evaluation of the 1RNA agent ίη νίνο can be facilitated by using a 1RNA agent conjugated to a detectable label (e.g., a fluorescent label, such fluorescein; a radioactive label, such as ³⁵ §, ³² P, ³³ P or ³ N; gold particles or antigen particles for immunohistochemistry) or other suitable method of determination.

The 1RNA agent can be evaluated for its ability to negatively regulate viral gene expression. The expression levels of the viral gene ίη νίνο can be determined, for example, by hybridization of ίη 8Jc or by isolating RNA from the tissue before and after exposure to the 1RNA agent. In cases where animals must be sacrificed to obtain tissue, a control animal that has not been exposed will serve as a comparison. The viral target mRNA can be detected by any desired method, including, but not limited to, RT-RCR, Northern blotting, a branched DNA method or an analysis of protection against RNase. Alternatively or additionally, viral gene expression can be controlled by Western blotting on tissue extracts treated with the 1RNA agent, or with E18L. Virus titer can be determined using plaque forming units.

- 7 012573

RNA Chemistry 1.

Here, isolated 1RNA agents are described, for example, BK RNA agents that mediate RNA1 to inhibit the expression of a viral gene, for example, P protein B8U.

The RNA agents discussed herein include other unmodified RNAs, as well as RNAs that have been modified, for example, to improve performance, and nucleoside substitute polymers. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugar, base and phosphate moieties, are the same or essentially the same as those found in nature, preferably that naturally occur in the human body. Rare or unusual, but naturally occurring RNAs in the form of modified RNAs have been mentioned in this area, see, for example, Lybac c1 a1. (1994), Νιιοίοίο Bs1bq Century. 22: 2183-2196. Such rare or unusual RNAs, often called modified RNAs (apparently due to the fact that they are the result of post-transcriptional modification), when used here, are included in the concept of unmodified RNA. The modified RNA as used herein refers to a molecule in which one or more of the nucleic acid components, namely sugars, bases and phosphate moieties, are different from those found in nature, preferably different from those found in the human body. Although they are called modified “RNAs,” they, of course, due to modification will include molecules that are not RNAs. Nucleoside substitutes are molecules in which the ribophosphate backbone is replaced by a non-ribophosphate construct, which allows the bases to be in the correct spatial arrangement, so that the hybridization is essentially similar to the hybridization observed with the ribophosphate skeleton, for example, uncharged materials that mimic the ribophosphate skeleton. Here are examples of all of the above.

The modifications described herein can be made to any double-stranded RNA and the RNA-like molecule described herein, for example, an 1RNA agent. It may be desirable to modify one or both of the antisense and sense strands of the 1RNA agent. Since nucleic acids are polymers of subunits or monomers, many of the modifications described below are found at the position that is repeated in the nucleic acid, for example, a modification of a base or phosphate moiety or non-binding O phosphate moiety. In some cases, the modification will occur in nucleic acid in all positions exposed, but in many, and in most cases, in reality, this will not happen. For example, modification can only occur at the 3'- or 5'-terminal position, can only occur in the terminal region, for example at the position on the terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modification can occur in a double-helix section, a single-spiral section, or both. For example, a phosphorothioate modification in the position of non-binding O can occur only at one or both ends, can occur only in terminal regions, for example, in the position on the terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of the chain, or it can occur in double helix and single helix areas, especially at the ends. Similarly, modification can occur in the sense chain, antisense chain, or both. In some cases, the sense and antisense chains will have the same modifications or modifications of the same class, and in other cases the sense and antisense chains will have different modifications, for example, in some cases it may be desirable to modify only one chain, for example, the sense chain.

The two main tasks for introducing modifications to 1RNA agents are stabilization with respect to cleavage in the biological environment and improvement of pharmacological properties, for example, pharmacodynamic properties, which are further discussed below. Other suitable modifications to the sugar, base or skeleton of the 1RNA agent are described in the joint application PCT / I82004 / 01193, filed January 16, 2004. The 1RNA agent may include a non-natural base, for example, the bases described in the joint application PCT / I82004 / 011822, filed April 16, 2004. The 1RNA agent may include unnatural sugar, for example a non-carbohydrate cyclic carrier molecule. The characteristic features of non-natural sugars for use in 1RNA agents are described in joint application PCT / I82004 / 11829, filed April 16, 2003.

The 1RNA agent may contain an internucleotide linkage (e.g., a chiral phosphorothioate linkage) suitable for enhancing resistance to the action of nuclease. Additionally or alternatively, the 1RNA agent may include a ribose-like compound for enhanced nuclease resistance. Typical internucleotide bonds and ribose-like substances for increased resistance to nuclease are described in joint application PCT / I82004 / 07070, filed March 8, 2004.

The 1RNA agent may include ligand conjugated monomeric subunits and monomers for oligonucleotide synthesis. Typical monomers are described in joint application US No. 10/916185, filed August 10, 2004

1RNA agent may have a ΖΧΥ-structure, for example, described in the joint application PCT / I82004 / 07070, filed March 8, 2004

The 1RNA agent can complex with the amipipathic moiety. Typical amphipathic parts for use with 1RNA agents are described in joint application PCT / I82004 / 07070, published March 8, 2004.

In another embodiment, the 1RNA agent may be formulated into a complex delivery substance, the feature of which is a modular complex. This complex may include a carrier associated with one or more (preferably two or more, more preferably all three): (a) a condensing agent (for example, a substance capable of attracting, for example, binding, nucleic acid, for example, by ionic or electrostatic interactions); (B) a fusion agent (for example, a substance capable of merging and / or transported through the cell membrane) and (c) a leading group, for example, an agent that induces a lectin, glycoprotein, lipid or protein on the cell or tissue, for example, an antibody that binds to the target type of cell. 1RNA agents formulated into a complex delivery agent are described in PCT / i82004 / 07070, filed March 8, 2004.

The 1RNA agent may have noncanonical pairings, for example, between the sense and antisense sequences of the RNA1 duplex. Characteristic features of noncanonical 1RNA agents are described in joint application PCT / I82004 / 07070, filed March 8, 2004.

Increased resistance to nuclease.

1RNA agent, for example, 1RNA agent that targets K.8U. may have increased resistance to nucleases.

For increased resistance to nuclease and / or affinity of binding to the target, the 1RNA agent, for example, the sense and / or antisense strand of the 1RNA agent, may contain, for example, 2'-modified ribose units and / or phosphorothioate bonds. For example, the 2 'hydroxyl group (OH) may be modified or replaced by a number of different “hydroxy” or “deoxy” substituents.

Examples of modifications of the “oxy” -2 ′ hydroxyl group include alkoxy or aryloxy (OK, for example, K = H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycols (PEG), O (CH ₂ CH ₂ O) _P CH ₂ CH ₂ OK; "Closed" nucleic acids (ΕΝΑ), in which 2 'hydroxyl is connected, for example, by a methylene bridge, with 4' carbon of the same ribose; O-amine and aminoalkoxy, O (CH ₂ ) _p- amine (e.g. amine = NH ₂ ; alkylamino, dialkylamino, heterocyclylamino, arylamino, diarylamino, heteroarylamino or diheteroarylamino, ethylenediamine, polyamino). It is noteworthy that oligonucleotides containing only a methoxyethyl group (MOE) (OCH ₂ CH ₂ OCH ₃ , PEG derivatives) exhibit nuclease resistance comparable to that of oligonucleotides modified with a robust phosphorothioate modification.

“Deoxy” modifications include hydrogen (ie, deoxyribose sugars, which are especially relevant to the protruding parts of partially RNA); halogen (e.g. fluorine); amino (e.g. ΝΠ ₂ , alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino or amino acid); NΠ (CH ₂ CH ₂ NΠ) _η CH ₂ CH ₂ - amine (amine = NΠ ₂ , alkylamino, dialkylamino, heterocyclylamino, arylamino, diarylamino, heteroarylamino or diheteroarylamino), -NΠC (O) Κ (K = alkyl, cycloalkyl, aryl , aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl thioalkyl; thioalkoxy and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may optionally be replaced, for example, with a functional amino group.

Preferred substituents are 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl, 2'-C-allyl and 2'-fluoro.

One way to increase resistance is to identify cleavage sites and modify such sites to inhibit cleavage, as described in US Joint Application No. 60 / 559,917, filed May 4, 2004. For example, 5'-IA3 ', 5'-iO-3' dinucleotides, 5'-CA-3 ', 5'-II-3' or 5'-CC-3 'may serve as cleavage sites. Therefore, increased resistance to nuclease can be achieved by modifying the 5 'nucleotide, resulting, for example, at least one 5'-uridine-adenine-3' (5'-IA-3 ') dinucleotide, where uridine is 2 '-modified nucleotide; at least one 5'-uridine-guanine-3 '(5'-IO-3') dinucleotide, where 5'-uridine is a 2'-modified nucleotide; at least one 5'-cytidinadenin-3 '(5'-CA-3') dinucleotide, where 5'-cytidine is a 2'-modified nucleotide; at least one 5'-uridine-uridin-3 '(5'-ii-3') dinucleotide, where 5'-uridine is a 2'-modified nucleotide; or at least one 5'-cytidine-cytidin-3 '(5'-CC-3') dinucleotide, where 5'-cytidine is a 2'-modified nucleotide. The 1RNA agent may include at least 2, at least 3, at least 4, or at least 5 such dinucleotides. In some embodiments, all pyrimidines of the 1RNA agent carry a 2'-modification, and therefore, the 1RNA agent has increased resistance to endonucleases.

To maximize nuclease resistance 2 ′ modifications can be used in combination with one or more phosphate linker modifications (eg, phosphorothioate). The so-called "chimeric" oligonucleotides are nucleotides containing two or more different modifications.

The incorporation of sugars in furanose form into the oligonucleotide skeleton can also reduce endonucleolytic cleavage. 1RNA agent can optionally be modified by the inclusion of 3 '

- 9 012573 cationic group or by inverting the nucleoside at the 3'-end of the 3'-3 'bond. In another embodiment, the 3'-end may be blocked by an aminoalkyl group, for example 3'C5-aminoalkyl bT. Other 3 ′ conjugates may inhibit 3′-5 ′ exonucleolytic cleavage. Without being bound by theory, a 3 ′ conjugate, such as naproxen or ibuprofen, can inhibit exonucleolytic cleavage by sterically blocking exonuclease from binding to the 3 ′ end of the oligonucleotide. Even small alkyl chains, aryl groups or heterocyclic conjugates or modified sugars (Ό-ribose, deoxyribose, glucose, etc.) can block 3'-5'-exonucleases.

Similarly, 5 ′ conjugates can inhibit 5′-3 ′ exonucleolytic cleavage. Without being bound by theory, a 5 ′ conjugate, such as naproxen or ibuprofen, can inhibit exonucleolytic cleavage by sterically blocking exonuclease from binding to the 5 ′ end of the oligonucleotide. Even small alkyl chains, aryl groups or heterocyclic conjugates or modified sugars (Ό-ribose, deoxyribose, glucose, etc.) can block 3'-5'-exonucleases.

The 1RNA agent may have increased resistance to nucleases in cases where the double-stranded 1RNA agent includes a single-stranded nucleotide protrusion at least at one end. In preferred embodiments, the implementation of the nucleotide protrusion includes from 1 to 4, preferably from 2 to 3 unpaired nucleotides. In a preferred embodiment, the unpaired nucleotide of a single-stranded protrusion that directly adjoins the terminal pair of nucleotides contains a purine base, and the terminal pair of nucleotides is a C-C pair or at least two of the last four complementary nucleotide pairs are C-C pairs. In further embodiments, the nucleotide protrusion may have 1 or 2 unpaired nucleotides, and in an exemplary embodiment, the nucleotide protrusion is 5'-CC-3 '. In preferred embodiments, the nucleotide protrusion is at the 3 ′ end of the antisense strand. In one embodiment, the 1RNA agent includes a 5'-CCC-3 'motif at the 3'-end of the antisense strand so that a 2-nucleotide protrusion of 5'-CC-3' is formed.

Thus, the 1RNA agent may include modifications so as to inhibit degradation, for example, by nucleases, for example endonucleases or exonucleases located in the patient’s body. These monomers are called ΝΚΜ here, or monomers stimulating resistance to nuclease, the corresponding modifications are ΝΚΜ-modifications. In many cases, these modifications will also modulate other properties of the 1RNA agent, for example, the ability to interact with a protein, for example, a transport protein, for example, serum albumin or a member of K1BS, or the ability of the first and second sequences to form a duplex with another sequence, for example, a target molecule.

One or more ΝΚΜ modifications can be introduced into a 1RNA agent or a sequence of a 1RNA agent. ΝΚΜ-modification can be used more than once in a sequence or in a 1RNA agent.

ΝΚΜ-modifications include some modifications that can only be located at the ends, and others that can follow in any position. Some модификации-modifications that can inhibit hybridization are preferably used only in the terminal regions and more preferably not in the cleavage site or in the cleavage region of a sequence that targets the sequence or gene, especially the antisense strand. They can be used everywhere in the sense strand provided that sufficient hybridization is maintained between the two strands of the bk 1RNA agent. In some embodiments, it is desirable to place ΝΚΜ in the cleavage site or in the cleavage region of the sense strand, since this can minimize inappropriate suppression.

In most cases, ΝΚΜ-modifications will be distributed differently depending on whether they are contained in a sense or antisense chain. On the antisense strand, modifications that inhibit or inhibit endonuclease cleavage should not be inserted into the region that undergoes HSC-mediated cleavage, such as a cleavage site or cleavage region (as described in E1bak1 c1 a1., 2001, Sepec apb. 15: 188, included here as a reference). Cleavage of the target occurs approximately in the middle of 20 and 21 nucleotides of the antisense strand, or about 10 or 11 nucleotides above the first nucleotide on the target mRNA, which is complementary to the antisense strand. The cleavage site used here refers to nucleotides either on the side of the cleavage site on the target mRNA or on the 1RNA agent chain that hybridizes with it. The cleavage region means nucleotides within 1, 2 or 3 nucleotides of the cleavage site, in both directions.

Such modifications can be introduced in the end regions, for example, in the end position or within the 2, 3, 4 or 5 positions of this end, the sequence that targets the effect, or the sequence that the target does not affect the patient sequence.

Bound ligands.

The properties of the 1RNA agent, including its pharmacological properties, can be influenced or can be set, for example, by introducing ligands, for example, attached ligands.

- 10 012573

A wide variety of substances, for example, ligands, for example, to a carrier of a subunit of a monomer conjugated to a ligand, can be bound to the 1RNA agent. Examples are described below in relation to a monomeric subunit conjugated to a ligand, but it is only preferred; the substances can be combined with the 1RNA agent in other places.

Preferred ligands are ligands that are connected, preferably covalently, either directly or indirectly via an intermediate bond, to a carrier. In preferred embodiments, the ligand is attached to the carrier via an intermediate bond. A ligand, or an attached ligand, may be present on the ligand-conjugated monomer when the ligand-conjugated monomer is integrated into the growing chain. In some embodiments, the ligand may be incorporated into the “precursor” of the ligand-conjugated monomer subunit after the “precursor” of the ligand-conjugated monomer subunit has been inserted into the growing chain. For example, a monomer having, for example, an amino-terminal bond, for example TAP- (CH ₂ ) pN2, can be integrated into a growing sense or antisense chain. In the subsequent process, i.e. after embedding the monomer subunit precursor in a chain, a ligand having an electrophilic group, for example a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the ligand-conjugated monomer precursor by attaching an electrophilic ligand group to the terminal nucleophilic bond group of the monomer subunit conjugate precursor ligand.

In preferred embodiments, the ligand modifies the distribution, targeting, or lifetime of the 1RNA agent into which it is inserted. In preferred embodiments, the implementation of the ligand provides increased affinity for the selected target, for example a molecule, cell or cell type, compartment, for example a compartment of a cell or organism, tissue, organ or area of the body, for example, compared with species that do not have such a ligand.

Preferred ligands can improve transport, hybridization and specific properties, and can also improve the nuclease resistance of the resulting natural or modified oligonucleotide or polymer molecule containing only a combination of the monomers and / or natural or modified ribonucleotides described herein.

In general, ligands may include therapeutic modifiers, for example, to enhance uptake; diagnostic compounds or reporter groups, for example, to monitor distribution; cross-linking agents; parts that confer resistance to nuclease; and natural or unnatural nucleic bases. Common examples include lipophilic molecules, lipids, lectins, steroids (e.g. uvol, hecigenin, diosgenin), terpenes (e.g. triterpenes, e.g. sarsasapogenin, Friedelin, epipriedelanol derivative of lithocholic acid), vitamins, carbohydrates (e.g. dextran, poole chitin, chitosan, inulin, cyclodextrin or hyaluronic acid), proteins, protein binding substances, integrin targeting molecules, polycations, peptides, polyamines and peptidomimetics.

The ligand may be a natural, or recombinant, or synthetic molecule, such as a synthetic polymer, for example a synthetic polyamino acid. Examples of polyamino acids include polyamino acid polylysine (Pb), poly-b-aspartic acid, poly-b-glutamic acid, a styrene-maleic anhydride copolymer, a poly (b-lactide-co-glycolide) copolymer, a maleic divinyl ester and maleic divide ester N- (2-hydroxypropyl) methacrylamide (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (RUA), polyurethane, poly (2-ethyl acrylic acid), polymers Ν-isopropyl acrylamide or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (Pb), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptide-like polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic moieties, for example cationic lipid, cationic porphyrin, quaternary salt of polyamine or alpha helical peptide.

Ligands may also include guidance groups, for example, agents that target a cell or tissue, for example thyrotropin, melanotropin, surfactant protein A, mucin, glycosylated polyamino acid, transferrin, bisphosphonate, polyglutamate, polyaspartate or ΚΟΌ peptide or ΚΟΌ like peptide.

Ligands can be proteins, for example, glycoproteins, lipoproteins, for example low density lipoproteins (LDL), or albumin, for example human serum albumin (HSA), or peptides, for example molecules with specific affinity for coligands, or antibodies, for example, an antibody that binds to a given type of cell, for example, malignant cells or osteocytes. Ligands may also include hormones and hormone receptors. They may also include non-peptide species such as cofactors, multivalent lactose, multivalent galactose, Ν-acetylgalactosamine, Ν-acetylglucosamine, multivalent manose or multivalent fucose. The ligand may be, for example, a lipopolysaccharide, a p38 MAP kinase activator or a ΝΓ-κΒ activator.

The ligand can be a substance, for example, a medicine, which can enhance the uptake of a 1RNA agent into a cell, for example, by destroying a cellular cytoskeleton, for example, by destroying cellular microtubules, microfilaments and / or intermediate filaments. This le

- 11 012573 the means of choice may be, for example, taxon, vincristine, vinblastine, cytochalazine, nocodazole, jasplacinolide, latrunculin A, phalloidin, swine solid A, indanocin or myoservin.

In one aspect, the ligand is a lipid or lipid based molecule. Such a lipid or lipid-based molecule is preferably bound to a whey protein, for example human serum albumin (HAC). Other molecules that can bind to HSA can also be used as ligands. For example, neproxin or aspirin may be used. A lipid ligand or lipid-based ligand can (a) increase the resistance to degradation of the conjugate, (b) improve the guidance or increase transport to the target cell or cell membrane, and / or (c) can be used to regulate binding to a serum protein, for example, HSA .

A lipid-based ligand can be used to modulate, for example, regulate the binding of a conjugate to a target tissue. For example, a lipid-based lipid or ligand that binds to HSA more strongly is less likely to be sent to the kidneys and, therefore, is less likely to be excreted from the body. A lipid ligand or a lipid-based ligand that is less strongly bound to HSA can be used to direct this conjugate to the kidneys.

In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity, so that this conjugate will preferably be distributed into non-renal tissue. However, it is preferred that the affinity is not so high that the binding of the HSA ligand is irreversible.

In another aspect, the ligand is a substance, such as a vitamin or nutrient, that is absorbed by a target cell, such as a proliferating cell. They are particularly useful in the treatment of diseases characterized by unwanted cell proliferation, for example, a malignant or non-malignant type, for example, malignant cells. Examples of vitamins include vitamin A, E, and K. Other example vitamins include group B vitamins, for example folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients absorbed by malignant cells.

In another aspect, the ligand is a substance that penetrates the cell, preferably a spiral substance that penetrates the cell. Preferably, this substance is amphipathic. A typical example is a peptide such as (a! Or ap1eppareE1a. If this agent is a protein, it can be modified, including peptidyl-like, inverters, non-peptide or pseudopeptide bonds and the use of Ό-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and lipophobic phase.

Modifications of 5'-phosphate.

In preferred embodiments, the tRNA agents are 5'-phosphorylated or include a phosphoryl analogue at the beginning of the 5'-end. Modifications of the 5'-phosphate antisense chain include modifications that are compatible with K18C-mediated gene suppression. Suitable modifications include 5'-monophosphate ((HO) ₂ (O) P-O-5 ');5'-diphosphate ((HO) ₂ (O) P-OR (HO) (O) -O-5 ');5'-triphosphate ((HO) ₂ (O) P-O- (HO) (O) P-O-P (HO) (O) -O-5 ');5'-guanosine cap (7methylated or unmethylated) (7t-С-О-5 '- (НО) (О) Р-О- (НО) (О) Р-О-Р (НО) (О) -О- 5');5'-adenosine cap (Arrr) and any modified or unmodified nucleotide cap structure. Other suitable modifications of 5'-phosphate will be known to those skilled in the art.

The sense strand can be modified to inactivate the sense strand and prevent the formation of active K18C, thereby potentially reducing non-target effects. This can be accomplished by modification, which prevents 5'-phosphorylation of the sense strand, for example, by modification with 5'-O-methylribonucleotide (see Yukapep e! A1. (2001) ATP hyg. | ΚΝΑ 1p1etGetepsera! Ceu. Ce11 107, 309-321.) Other modifications that prevent phosphorylation can also be used, for example, the simple substitution of 5'-OH with H rather than O-Me. Alternatively, a large bulky group can be added to 5 '-phosphate, converting it to a phosphodiester bond.

Delivery of 1RNA agents to tissues and cells.

Compositions.

The 1RNA agents described herein can be formulated for administration to a patient, preferably for local administration into the lungs and nasal passage (respiratory tissue) by inhalation or intranasal administration, or parenterally, for example by injection.

To simplify the presentation of the compositions, compositions and methods in this section are considered mainly in relation to unmodified tRNA agents. However, it should be clear that these compositions, compositions and methods can be used in practice with other 1RNA agents, for example modified 1RNA agents, and such use is included in the scope of this invention.

The formulated tRNA agent composition can take a number of forms. In some examples, the composition is at least partially crystalline, uniformly crystalline and / or anhydrous (for example, less than 80, 50, 30, 20, or 10% water). In another example, the 1RNA agent is in the aqueous phase, for example in a solution that contains water, this form is preferred for

- 12 012573 administration by inhalation.

Compositions in the aqueous phase or crystalline compositions can be included in the composition of the carrier, for example liposomes (especially for the aqueous phase) or particles (for example, microparticles suitable for the crystalline composition). Basically, the composition of the 1RNA agent is formulated so that it is compatible with the intended route of administration.

The preparation of the 1RNA agent can be made in combination with another agent, for example, another therapeutic agent or a means for stabilizing the 1RNA agent, for example, a protein that forms a complex with the 1RNA agent to form 1RNA. Other agents include chelating agents, for example, ΕΌΤΆ (for example, to remove divalent cations such as MD ²⁺ ), salts, RNase inhibitors (for example, a broad specificity RNase inhibitor such as RNAzin) and the like.

In one embodiment, the preparation of the 1RNA agent includes another 1RNA agent, for example, a second 1RNA agent that can mediate RNA1 in relation to the second gene. Other preparations may include at least three, five, ten, twenty, fifty or one hundred or more different types of RNA1. In some embodiments, these agents target the same virus but have different target sequences. In another embodiment, each 1RNA agent is directed to different viruses. As shown in the example, more than one virus can be inhibited by co-administration of two 1RNA agents simultaneously or at close time intervals, each of which is directed to one of the viruses being treated.

Methods of treatment and delivery routes.

A composition that includes a 1RNA agent of the present invention, for example, a 1RNA agent directed to an ultrasound, can be administered to a patient in various ways. Representative examples of routes of administration include inhalation, the intravenous route, nasal or oral drug delivery. A preferred method for administering 1RNA agents of the present invention is direct administration to the lungs and nasal passage, or systemic administration by parenteral administration.

The 1RNA agent may be included in pharmaceutical compositions suitable for administration. For example, the compositions may include one or more 1RNA agents and a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” means the inclusion of any or all solvents, dispersion medium, coatings, antibacterial and antifungal agents, isotonic substances and absorption retarding substances, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Since not any conventional medium or agent is compatible with the active compound, their use is contemplated in these compositions. Additional active compounds may also be included in these compositions.

The pharmaceutical compositions of the present invention can be administered in various ways, depending on which treatment is desired, topical or systemic, and depending on the area in which the treatment is carried out. Administration may be local (including intranasal or intrapulmonary), oral or parenteral. A preferred route of administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection.

Basically, the delivery of 1RNA agents of the present invention is carried out to reach the site of infection in the patient. A preferred way to achieve this is either by local administration into the lungs or nasal passage, for example into respiratory tissues by inhalation, nebulization or intranasal administration, or by systemic administration, for example parenteral administration.

Compositions for inhalation or parenteral administration are well known in the art. Such formulations may include sterile aqueous solutions, which may also contain buffers, diluents and other suitable additives, for example, PBH or 5% dextrose in water. For intravenous use, the total concentration of the solution should be adjusted to ensure the isotonicity of the drug.

The active compounds described herein are preferably administered to the patient’s lungs or nasal passages in any suitable manner. Active compounds can be administered by administering an aerosol suspension of respiratory particles containing the active compound or active compounds that the patient inhales. The active compound can be sprayed in various forms, such as, but not limited to, dry powder inhalation agents, inhalers with a metering device, or a liquid / liquid suspension. Inhaled particles may be liquid or solid. The particles may optionally contain other therapeutic ingredients, such as amiloride, benzamyl or phenamyl, with a selected compound included in an amount effective to suppress reabsorption of water from the mucous secretions of the respiratory tract, as described in US patent No. 4501729.

A particular pharmaceutical composition may optionally be combined with a carrier to facilitate dispersion or transfer. A suitable carrier, such as sugar (i.e., dextrose, lactose, sucrose, trehalose, mannitol), may be mixed with the active compound or compounds in any

- 13 012573 suitable ratio (for example, in a ratio of 1: 1 by weight).

Particles containing the active compound for the practical use of the present invention should include particles suitable for inhalation, i.e., particles of a sufficiently small size to pass through the mouth or nose and larynx during inhalation and into the bronchi and alveoli of the lungs. Generally, particles of about 1 to 10 microns in size (more preferably about less than 5 microns in size) are suitable for inhalation. Particles of a non-natural inhalation size that are included in the aerosol tend to be deposited in the throat and swallowed, and the amount of non-respirable particles in the aerosol is preferably reduced to a minimum. For nasal administration, a particle size in the range of 10-500 μm is preferred to provide a delay in the nasal cavity.

Liquid pharmaceutical compositions of the active compound for aerosol preparation can be prepared by combining the active compound with a suitable carrier, such as sterile pyrogen-free water. The hypertonic saline solutions used to carry out the present invention are preferably sterile pyrogen-free solutions containing from 1 to 15 wt.% Of a physiologically acceptable salt and more preferably from 3 to 7 wt.% Of a physiologically acceptable salt.

Liquid particles aerosols containing the active compound can be prepared by any suitable method, such as, for example, a pressure spray jet or an ultrasonic spray. See, for example, US Pat. No. 4,501,729. Nebulizers are commercially available devices that convert solutions or suspensions of the active ingredient to therapeutic aerosol dust, either by accelerating the release of compressed gas, usually air or oxygen, through a narrow venturi outlet or by ultrasonic shaking. .

Suitable compositions for use in nebulizers consist of the active ingredient in a liquid carrier, the active ingredient contains up to 40% w / w. composition, but preferably less than 20% wt./wt. The carrier is usually water (and most preferably sterile pyrogen-free water) or a dilute aqueous alcoholic solution, preferably made isotonic, but can be hypertonic with body fluids by adding, for example, sodium chloride. Optional additives include preservatives if the formulation is not sterile, for example, methyl hydroxybenzoate, antioxidants, flavorings, essential oils, buffering agents and surfactants.

Particulate aerosols containing the active compound can be prepared in a similar manner with any aerosol generator of therapeutic particulate matter. Aerosol generators for administering solid drug particles to a patient produce respirable particles and create an aerosol volume containing a predetermined measured dose of a therapeutic agent in an amount suitable for administration to a human. One illustrative type of aerosol particulate generator is an insufflator. Suitable formulations for administration by injection include finely divided powders that can be delivered using an insufflator or delivered to the nasal cavity by inhalation through the nose. In the insufflator, the powder (for example, its measured dose, effective for the treatment described here) is contained in capsules or cartridges, usually made of gelatin or plastic, which are either punctured or open ίη к1! And, and the powder is delivered by the expelled air through this device by inhalation or by hand pump. The powder used in the insufflator consists of either only the active ingredient or a powder mixture containing the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient usually contains from 0.1 to 100 wt./wt. composition.

A second type of illustrative aerosol generator includes an inhaler with a metering device. Metered-dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution of the active ingredient in a liquefied propellant. In use, these devices disperse the composition through a valve adapted to deliver a metered volume, typically from 10 to 200 μl, to produce a spray solution of fine particles containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and mixtures thereof. The composition may further comprise one or more cosolvents, for example ethanol, surfactants such as oleic acid or sorbitan trioleate, an antioxidant and suitable flavoring agents.

The introduction may be carried out by a patient or another person, for example, caring for the patient. A caregiver can be any person associated with providing care for a person, for example, in a hospital, hospice, doctor’s office, outpatient clinic; a healthcare professional, such as a doctor, nurse, or other practitioner; spouse or guardian, such as parents. Treatment may be with metered doses or with a dispenser delivering a metered dose.

- 14 012573

The term “therapeutically effective amount” is an amount. present in the composition. which is necessary to provide the desired amount of drug to the patient. who are being treated. to obtain the expected physiological response. In one embodiment, therapeutically effective amounts of two or more 1RNA agents. each one targets a different respiratory virus. e.g. K.8U. administered to the patient at the same time.

The term “physiologically effective amount” is an amount. delivered to the patient to obtain the desired prophylactic or therapeutic effect.

The term "pharmaceutically acceptable carrier" means. that the carrier can enter the lungs. without significant adverse toxicological effects on the lungs.

The term "co-administration" refers to the administration of two or more agents to a patient. in particular. two or more 1RNA agents. These agents may be contained in a separate pharmaceutical composition and may be administered at the same time, or these agents may be contained in separate compositions and may be administered to a patient sequentially. Because two agents can be detected in a patient at the same time. they say. that these two agents are introduced together.

Types of pharmaceutical excipients. which are used as a carrier. include stabilizers. such as human serum albumin (HSA); fillers. such as carbohydrates. amino acids and polypeptides; pH adjusters or buffers; salt. such as sodium chloride; and the like. These carriers may be in crystalline or amorphous form or may be a mixture of two forms.

Fillers. which are especially significant. include compatible carbohydrates. polypeptides. amino acids or combinations thereof. Suitable carbohydrates include monosaccharides. such as galactose. Ό-mannose. sorbose and the like; disaccharides. such as lactose. trehalose and the like; cyclodextrins. such as 2-hydroxypropyl-beta-cyclodextrin; and polysaccharides. such as raffinose. maltodextrins. dextrans, etc .; alditols. such as mannitol. xylitol, etc. A preferred carbohydrate group includes lactose. trehalose. raffinose. maltodextrins and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine. glycine is preferred.

Suitable pH adjusters or buffers include organic salts. derived from organic acids and bases. such as sodium citrate. sodium ascorbate and the like; sodium citrate is preferred.

Doses

1RNA agent can be administered in a single dose of about less than 75 mg / kg body weight or about less than 70. 60. 50. 40. 30. 20. 10. 5. 5. 2. 1. 0.5. 0.1. 0.05. 0.01. 0.005. 0.001 or 0.0005 mg / kg body weight and less than 200 nmol of 1RNA agent (for example, approximately 4.4x1016 copies) per 1 kg of body weight or less than 1500. 750. 300. 150. 75. 15. 7.5. 1.5. 0.75. 0.15. 0.075. 0.015. 0.0075. 0.0015. 0.00075. 0.00015 nmol of 1RNA agent per 1 kg of body weight. Single dose. eg. may be administered by inhalation or by spray or by injection. In one example, a dose in the range of 0.02-25 mg / kg is used.

The delivery of the 1RNA agent directly to the lungs or nasal passage can be in a dose of about 1 to about 150 mg / nasal passage.

The dose may be an amount. effective for treating or preventing a disease or disorder.

In one embodiment, a unit dose is administered once a day. In another use, a single dose is administered twice on the first day. and then daily. Alternatively. a single dose may be less than once a day. eg. less than every 2. 4. 8 or 30 days. In another embodiment, a unit dose is not administered at a specific frequency (e.g., irregular frequency). For example. a single dose can be administered only once. Since 1RNA-mediated gene suppression may persist for several days after administration of the 1RNA-agent composition. in many cases, it is possible to administer the composition with a frequency of less than once a day, or in some cases only once during the entire treatment period.

In one embodiment, the patient is administered an initial dose and one or more maintenance doses of an 1RNA agent. for example a double-stranded 1RNA agent. or an 81RNA agent (e.g., a precursor. for example, a larger 1RNA agent. which can be processed into an sRNA agent or DNA. which encodes a 1RNA agent. for example a double-stranded 1RNA agent or 81RNA agent or its precursor). The maintenance dose or doses are usually lower than the initial dose. for example, half the initial dose. The maintenance regimen may include treating a patient with a dose or doses in the range of 0.01 to 75 mg / kg body weight per day. e.g. 70. 60. 50. 40. 30. 20. 10. 5. 2. 1. 0.5. 0.1. 0.05. 0.01. 0.005. 0.001 or 0.0005 mg / kg body weight per day. Maintenance doses are preferably administered no more than once every 5-14 days. Besides. the treatment period may continue for a period of time. which will vary depending on the nature of the particular disease. its severity and general condition of the patient. In preferred embodiments, the dose can be administered no more than once a day. for example, no more than once every 24. 36. 48 hours or more. for example, no more than once every 5 or 8 days. After treatment, changes are monitored.

- 15 012573 patient and relieving symptoms of a painful condition. The dose of the compound can either be increased if the patient does not have a noticeable reaction to the current dose levels, or the dose can be reduced if the symptoms of the disease state are noticeable, the disease state has been withdrawn, or if undesirable side effects are observed.

In one embodiment, the pharmaceutical composition of the 1RNA agent includes many types of 1RNA agents. Varieties of 1RNA agents may have sequences that do not overlap and are not adjacent to natural target sequences, for example, the target sequence of the K.8U gene. In another embodiment, many types of 1RNA agent are specific for various natural target genes. For example, a 1RNA agent that targets the B8U protein P gene may be present in the same pharmaceutical composition as a 1RNA agent that targets another gene, such as the N protein gene. In another embodiment, 1RNA agents are specific for different viruses, for example K.8U.

The concentration of the composition of the 1RNA agent is an amount effective enough to treat or prevent a disease or to regulate a physiological state in humans. The concentration or amount of the administered 1RNA agent will depend on the parameters determined for that agent and the route of administration, for example, nasal or pulmonary. For example, nasal formulations require much lower concentrations of certain ingredients to avoid irritation or burns to the nasal passages. Sometimes it is necessary to dilute the oral composition up to 10-100 times to obtain a suitable composition for nasal administration.

Certain factors can affect the dose needed to effectively treat a patient, including, but not limited to, the severity of the disease or disorder, previous treatment, the general health status and / or age of the patient, and the presence of other diseases. It will also be understood that an effective dose of a 1RNA agent, such as a 51RNA agent used for treatment, may increase or decrease over the course of a particular treatment. Changes in the dose may be due to or become apparent from the results of diagnostic tests. For example, a patient is observed after administration of a 1RNA agent composition. Based on the observation information, an additional amount of the 1RNA agent composition may be added.

The invention is further illustrated by the following examples, which are not limiting.

Examples

Creation of antiviral 51RNAs against K.8U mRNA.

51RNA against P, N and L K.8U mRNA was synthesized chemically using known methods. The 51RNA sequences and some inhibition of cross activity between the subtypes and the 1C ₅₀ values are listed (Table 1 (a-c)).

Research ίη νίίΓΟ and viral infection.

Uego cells E6 were cultured to 80% confluence in ΌΜΕΜ containing 10% EB8 inactivated by heat. To introduce 51RNA, 4 μl of TgapSCH-TKO was added to 50 μl of serum-free ΌΜΕΜ and incubated at room temperature for 10 min. Then, the indicated concentration of 51RNA was added to the medium / TCR reagent, respectively, and incubated at room temperature for 10 min. An RNA mixture was added to 200 μl ΌΜΕΜ containing 10% EB8, and then to the cell monolayer. Cells were incubated at 37 ° C, 5% CO ₂ for 6 hours. The RNA mixture was removed by careful washing with 1x Hanks balanced salt solution (HB88), 300 plaque-forming units (p £ and) B8U / L2 (ΜΟΙ = 30) were added to the wells well and subjected to adsorption for 1 h at 37 ° C, 5% CO ₂ . The virus was removed and the cells were washed with 1x HB88. Cells were coated with 1% methylcellulose in содержащей containing 10% EB8 medium and incubated for 6 days at 37 ° C, 5% CO ₂ . Cells were immunostained for plaques using monoclonal antibody 131-2A to E protein.

Delivery of 51 RNA and viral infection ίη νίνο.

Pathogen-free female VLV / s mice aged 4 weeks were purchased from Nag1ap. Mice were anesthetized during infection and intranasal drip (ί.η.). The mice were immunized by intranasal drip of the indicated amount of 51RNA, either unbound or in combination with 5 μl Tgapky TKO. 150 μl of 8 fall15 (monoclonal antibody clone 143-6C, to K.8U E protein) and the mouse isotype control (1dO1) were administered intraperitoneally (ί.ρ.) 4 hours before K.8U infection (10 ⁶ REI K.8U / L2 ) Used 10 mice per group. The mass of animals was checked at 0, 2, 4 and 6 days after infection. Lungs were taken on day 6 after infection and examined on K.8U using the analysis of immunological staining of plaques.

Analysis of immunological staining of plaques.

24-well plates with Uego cells E6 were cultured up to 90% confluence in ΌΜΕΜ containing 10% EB8, heat inactivated. The lungs of mice were homogenized with a manual homogenizer in 1 ml of sterile PB8 Dulbecco (Ό-PBZ) and diluted 10 times with serum-free ΌΜΕΜ. Dilutions of the lysate of the lung tissue containing the virus were placed in 24-well plates in triplicate and adsorbed for 5 hours at 37 ° C 5% CO ₂ . Wells were coated with 1% methylcellulose in ΌΜΕΜ,

- 16 012573 containing 10% PB8. Then the plates were incubated for 6 days at 37 ° C, 5% CO ₂ . After 6 days, the integument was removed and the cells were fixed in acetone: methanol (60:40) for 15 minutes. Cells were blocked with 5% milk powder in PB8 (Mik / PB8) for 1 h at 37 ° C. An antibody to R protein of CBU (131-2A) was added to the wells at a dilution of 1: 500 and incubated for 2 hours at 37 ° C. Cells were washed twice in PB8 / 0.5% Tetep 20. A complex of goat anti-mouse 1C with alkaline phosphatase at a dilution of 1: 500 was added to the wells and incubated for 1 h at 37 ° C. Cells were washed twice in PB8 / 0.5% Tbp 20. The reaction was developed using a set of Ul1og'5 A1k11pe Rk8pka! A8e 8b8! Ha! With kN II (Ues! Gg B1ask) and was stained with hematoxylin. Plaques were visualized and counted using an inverted O1ushri8 microscope.

Processing Analysis.

Mice were infected with CBU (10 ⁶ RRB CBU / A2) by intranasal drip on day 0 and 50 μg of the indicated 81RNA administered by intranasal drip were treated at the indicated times (day 1-4 after viral infection). 3-5 mice per group were used and titers of the virus in lung lysates were measured on day 5 after infection with the virus, as described previously.

Ιη Uygo inhibition of CBU using 1RNA agents.

1RNA-agens presented in table. 1 (a-c) were tested for anti-CBU activity in plaque assay as described above (FIG. 1). Each column (column) represents a 1RNA agent, (Table 1 (ac)), for example, column 1 represents the first agent of the table. 1a, the second column is a second agent, etc. Active 1RNA agents were identified by% of remaining virus. Some agents have been identified that show up to 90% inhibition. The results are summarized in table. 1 (a-s).

The ίη ugo inhibition of CBU was determined depending on the dose using 1RNA agents. Examples of active agents from the table. 1 were tested for anti-CBU activity in a plaque assay as described above at four concentrations. A dose-dependent response was found with the tested active 1RNA agents (Fig. 2) and is summarized in table. 1 (a-s).

Ιη ugo subtype inhibition In CBU using 1RNA agents was tested as described above. 1RNA agents of FIG. 2, tested for anti-CBU activity against subtype B (FIG. 3). Subtype CBU was inhibited by 1RNA agents tested to varying degrees and summarized in table. 1 (a-s).

Ιη y | yo inhibition of CBU using 1RNA agents.

Ιη νί \ Ό inhibition of CBU using AB1729 and AB1730 was tested as described above. The agents described in FIG. 4, tested for anti-CBU activity in a mouse model. 1RNA agents effectively reduced the titers of the virus ίη νί \ Ό and were more effective than the control antibody (MAb 143-6c, mouse 1dC1 AB, which is approved for the treatment of CBU).

AB1730 was tested for dose-dependent activity using the methods described above. These agents showed a dose-dependent response (Fig. 5).

1RNA agents showing ίη yugo activity were tested for anti-CBU activity of ίη у1уо, as described above. Some agents showed a decrease in virus titers> 41 odz with prophylactic administration (Fig. 6).

1RNA agents showing ίη ugo and / or ίη у1уо activity were tested for anti-CBU activity of ίη у1уо, as described in the processing protocol above. Some agents showed a decrease in titers of 2-31odz virus (Fig. 7) when administered 1-2 days after viral infection.

Sequencing of isolates along the length of the target sequence.

The way.

Growing isolates and RNA isolation.

Clinical isolates from patients infected with CBU were obtained from Baggu Apyeghop at CO C in Atlanta, Georgia (4 strains), and 1o11p Oeutsepho from the University of Tennessee, Memphis (15 strains). When grown in HEP-2, human epithelial cells (ATCC, Ca! # SSB-23), it was noted that 4 isolates from Georgia grew slower than 15 strains from Tennessee; therefore, they were processed and analyzed separately. This process is briefly described as follows.

Uego E6, monkey kidney epithelial cells (ATCC, Ca! # SVB-1586) were grown to 95% confluence and infected with primary isolates at a 1/10 dilution. The virus was absorbed for 1 h at 37 ° C, then the cells were supplemented with Ό-MEM and incubated at 37 ° C. Every day, the cells were checked for the presence of a cytopathic effect (CPE) using a light microscope. At 90% CPE, cells were harvested by scraping and pelleted by centrifugation at 3000 rpm for 10 minutes. RNA preparations were prepared according to standard methods in accordance with the manufacturer's instructions.

Amplification of the N gene of the KBU.

Viral RNA was collected after infection and used as templates in PCR using primers that hybridize above and below the target site AB-ER-2017 to amplify a fragment of ~ 450 nucleotide pairs. All RNA was denatured at 65 ° C for 5 min in the presence of direct and reverse primers for the N gene of CBU contained on ice, and then the reverse was performed

- 17 012573 transcription using 8ireg5epr1 ΙΙΙ (ΙηνίΙΐΌβοη) for 60 min at 55 ° C and for 15 min at 70 ° C. PCR products were analyzed by gel electrophoresis on a 1% agarose gel and analyzed according to standard protocols.

Results.

Sequencing of the first 15 isolates confirmed that the target site for L-OR-2017 was completely conserved in all strains. It is important that this conservatism persisted in all different populations, which included isolates of both subtype A and subtype B K8U. Interestingly, when analyzing 4 isolates growing more slowly, it was found that one of 4 (BAR6824) had a single base mutation in the recognition site L-OR-2017. This mutation changed the coding sequence at position 13 N of the K8U N gene in this isolate from A to C.

Conclusions.

From isolates from 19 patients, the K8U gene N sequence was determined in the target site for AB-OP-2017. In 18 of 19 cases (95%), the recognition element for L-ER-2017 is 100% conservative. One of the isolates had a single base change, changing the nucleotide at position 13 from A to C within N of the K8U gene. This change creates a single C: B ambiguous correspondence between the L-ER-2017 antisense chain and the target sequence. Based on considerations of the hybridization potential of such C: and ambiguous correspondence, it is assumed that L-ER-2017 will be effective in suppressing the K8U gene N in this isolate.

Isolation silencing data.

Ways.

Uego cells E6 were cultured to 80% confluence in ΌΜΕΜ containing heat inactivated 10% EB8. To introduce 81RNA, 4 μl of TganSCH-TKO was added to 50 μl of serum-free ΌΜΕΜ and incubated at room temperature for 10 min. Then, the indicated concentration of mRNA was added to the medium / TCR reagent, respectively, and incubated at room temperature for 10 minutes. A mixture of RNA was added to 200 μl ΌΜΕΜ containing 10% EB8, and then to the cell monolayer. Cells were incubated at 37 ° C, 5% CO ₂ for 6 hours. The RNA mixture was removed by careful washing with 1x Hanks balanced salt solution (HB88), 300 plaque-forming units (p! U) B8U / A2 (ΜΟΙ = 30) were added to the wells and subjected to adsorption for 1 h at 37 ° C, 5% CO ₂ . The virus was removed and the cells were washed with 1x HB88. Cells were coated with 1% methylcellulose in содержащей containing 10% EB8 medium and incubated for 6 days at 37 ° C, 5% CO ₂ . Cells were immunostained for plaques using a monoclonal antibody 131-2 A to E protein.

Results.

Silencing was observed in all isolates (table. 2)

Output.

All tested clinical isolates were specifically inhibited by 2017 cRNA by more than 85%. There were no isolates significantly inhibited by the action of 2153 mRNA, which was incompletely paired as a control.

Silencing in a plasmid-based study.

The way.

A 24-well plate was seeded with Heb 86 cells and grown to 80% fusion. For each well, 1 μg of K8U X-U5 plasmid was mixed with mRNA (under the indicated conditions) in 50 μl of PTI-ΜΕΜ and added to a mixture of 2000 C ^ Yogod-Orytet, prepared in accordance with the manufacturer's instructions, and left to stand for 20 min at room temperature for complex formation. The complex was added to the cells and incubated at 37 ° C. overnight. The medium was removed, the PB8 cells were washed and lysed using 50 μl of lysis buffer (K1RL buffer (50 mm TgC-HC1 pH 8.0, 150 mm No. C1, 1 mm No., 0.5% deoxycholate No. 1% No. 40, 0.05% 8–8) for 1-2 minutes Incubation was quantified by measuring the level of K8U protein in cell lysates, as determined by Western blotting with anti-Y5 antibody.

Results.

Transient plasmid expression has been shown to be an effective assay for 1RNA agents (Table 3).

Conclusions.

81RNA 2017 specifically and in a dose-dependent manner inhibits the production of N protein K8U N during transient cotransfection with a plasmid expressing the K8U gene N. Inhibition was not observed with uncompletely paired 2153 cRNA as a control.

Suppression of K8U by aerosol delivery of mRNA.

The way.

mg / ml of an AB-OR-1729 or AB-OR-1730 solution was delivered by spraying using an aerosol device for a total of 60 seconds. The virus was obtained from the lungs, as described above, and analyzed using ΕΌΙ8Α rather than plaque assay. In ΕΌΙ8Α, the concentration of N protein K8U in the cells infected with the virus obtained from mouse lysates was determined.

- 18 012573

EB18A.

The lung lysate was diluted 1: 1 with carbonate-bicarbonate buffer (ΝαΗΟΘ ₃ pH 9.6) to a working concentration of 6-10 μg / 100 μl, added to each test well and incubated at 37 ° C for 1 h or overnight at 4 ° C. The wells were washed with 3 × PB8 / 0.5% T1 + 20, then blocked with 5% milk powder / PB8 for 1 h at 37 ° C or overnight at 4 ° C. Primary antibody was added to the wells (P protein positive control = clone 131-2A; O protein positive control = 130-2O; negative control = normal 1dO1 (WB RRagstdep, CaP # 553454, test serum or hybridoma supernatant) 1: 1000 and incubated at 37 ° C for 1 h or overnight at 4 ° C. The wells were washed with 3 × PB8 / 0.5% T% 20. Secondary antibody (whole goat anti-mouse 1dO (H + B) molecule conjugated to alkaline phosphatase) diluted 1: 1000, added to the wells (100 μl / well) and incubated at 37 ° C for 1 h or overnight at 4 ° C. washed with 3 × PB8 / 0.5% T% 20, then Νρρ (ΒίμιικιΕίδΓ) was added substrate 81 of AMPSR N2770 in accordance with the manufacturers instructions, 200 μl of substrate per well was added and incubated for 10-15 minutes Absorption was measured at ΘΌ 405/495.

Output.

The delivery of specific δί P11K K8U reduces the N levels of K8U protein in the lungs of mice compared to the control of an incompletely paired δί P11K (Fig. 8A, B).

Inhibition of ΐπ νίνο on day 3 is prevention.

The way.

Ιη νίνο prophylaxis was tested using the ΐπ νίνο method described above, except that δί P11K was delivered at various points in time before K8U infection from 3 days before infection to 4 hours before infection.

Results.

δί P11K, administered intranasally up to 3 days prior to viral infection, shows a significant suppression of ΐπ νίνο (Fig. 9).

Sequences δί P11K

Table 1a

B gene K8U

Fact start The starting point is USHEeas! Semantic 5ЕО Ш Шз Antisense 8EO I " Щ: AL-OR # % inhibition of KZZ / A2 (5nM) % inhibition of Ya5UA2 500 pM % inhibition of K5UA2 50 pM % inhibition of H5UA2 5 pM % inhibition of K8UV (5nM) 3 ( osaussia iiliil α υσσ datat one ISLA A IL A YOSS LISSATAT one ΑΕΌΡ-2024 92 4 2 bAisssAiiliiddioBalatat 3 IissAiilAilAisosAisatat 4 LYUR-Zhb 82 49 47 lsiliyiiAAAAOsioiiA (1T <yy 5 IlASASciiiiAAAiAAsiatat 6 AFFR-Shb fifty AB συυΑυυυΑΑΛΑοσυπυυΑυΰΤΰτ 7 AilASAseishiAADiAAsatat § AL-OR-2117 53 51 LiidAAAssisiiAisisotite nine SAOlilASlssiiii AAAAiatat 10 AB-2118 55 53 qiAllASsissiiisisshatat eleven AABLSAAASlssiiiilAatat 12 L-OR-2119 156 154 ALsissAsilsils ASSA . 12 AgiosiAsiAoiosAsiiatat 14 AE-TsR-2027 86 <57 155 ЛСиССАСиАСОСО АССААА4Т <ГГ fifteen IssissiAS-iAssissAsiatat sixteen ΑΕΌΡ-2028 90 158 156 oisSasilsyASloslidstat 12 AidissisiAsiAoibSasatat eighteen L-OR-2029 89 159 157 Islsi nineteen SAiliossisiAoiAbisolatate 22 AEOG 2030 86 341 339 OAAOAssiAiASLALiAAASYTOT eleven SiiliyisiAiAbsisiisatat 22 LZ.OG-212V 344 342 cssiAidoAAAIlAoioAotat 22 and USAAIiAiAbsiSatat 24 AB-2121 347 345 silyAOAldiAAsiadisiitat 25 ASAisAsiliyisiAiloatat 26 LIOR-2031 fifteen 554 552 IslALSAASLisiaSadetyt 27 IsaAbAbibiioiiiiioAatat 28 ΑΕΌΡ-2122 1004 1002 IloAssssAiiiAiilisisyTyt 29th OASliAlilAAississiAatat thirty AB-2123 1408 1406 diAAAASasyisiAAdil <1T <1T 31 AIIilbAAASssiiiiAiatat 32 ABOR-2124 1867 1865 SISDSIAIDSSIOAO Alioietat 32 ASAisiASilsilsioAbatsp 34 AL-TsR-2032 90 1868 1866 ISACisidsaiASldisishtat 35 AASliisilssiASASiolatat 2 " LE-OR-2033 84 1869 1867 SASIBIDSOYASAAAaiiistsp · 22 AAASAisiAssilsAsibatag 38 L-OG-2034 86 1870 1868 dsiailsoiASllisiisatat 29th SAAlsliisidssiASACASiatat thirty AB- »R-2Sh 1871 1869 sioilosiASAAipiiissatag but ass asa isilssi aslatsat 42 AL-OR-2113 1978 1976 lsAASAiAyssisAisilsatat 42 SIABLISCASidisiiioiatat 44 AL-OR-2035 89 2104 2102 LSSAALiiSALiSALSSASHTyt 43 lissium iodine OAAiiiosiatat 46 ΑΕ-ϋΡ-2036 87

- 19 012573

2103 2288 2382 2384 2385 2483 2485 2505 2506 IslalisAisAAOSlisht olshAASAllsiosAiiAstat iAliAisisisAllsosAbtot AidisisisALASSASALaiItisisisAlsssAaIlistht SlissisAlssASAiAliogsIssiSalisAliAssaliiIsaliiIssaliiIlii 47 49 51 53 55 52 59 & 63 AAAiosiyoAbISAiiiosatat AilAissAsiiiaiisAisetat IsissicibAlbAiidatat AiiisssiiibAOAlBaiAiatat AliasssiiibAOAbAiAatat AliAlisibsiiAbsliabiatiAibiati 42 52 52 54 56 52 22 62 64 AIOR-2037 Lb-OR-2038 Ab-OR-2125 ΑΖ-ΌΡ-Ζ126 Ab-ER-2127 Ab-OR-2039 Α6-Ι3Ρ-2040 AL-BR-2041 Ab-OR-2114 91 eleven 87 88 96 76 73 69 94 2509 2507 ssiillAilsssiilAliatat £ AiiiAloosiAiiiAAissatat 66 Ab-BR-2042 96 98 97 97 90 2510 2508 SliaAlliAOssiaAAlsmpgg 22 AabiiAAbbsiliaAAibatat 62 AL-CR-2043 97 86 79 75 94 2765 2763 IAIIAbsblobsbbAliiliyagat "2 aa or blAAsibSabAAbAvtat 22 ALOR-2044 97 79 72 67 84 2767 2765 Ilyssusiiilliyiiiiiatat 21 baalbabbaaaasibsabalatbt 72 AYUR-2045 fifteen 3283 3281 ALAAOIOSASAASAIililatate 22 IAIAAbbiisios AsiaBathat 74 Ab-PR-2128 .3284 3282 ALAOISLSAAAAIILILSATAT 25 bbAbaAbObbaibCASibiatat 22 Α6ΌΡ-2046 94 94 91 91 93 3338 3336 AillilsASSIASAIAissatsgg 22 ABABLIBBABOBBSBABABATABAT 78 Lb-BP-2047 87 3339 3337 IlloAlssilsleyAissilate 22 ASOAblbSbAObiisUAiAatat 22 Ab-PR-2048 84 3365 3363 iAlbAsiisiiilisAAloatat 81 SbbSLbaAAASAASbSbbAaTat 82 Ab-br-2129 4021 4019 lsACisAoilsilolssAiatat 83 fox bsb lei ASbb ASboiatat 84 Α6ΌΡ-2049 24 4022 4020 SACISILSILSIAOASCAISATAT 25 SAbObbSbASbASbbASboatat 22 AYUR-2050 fifteen 4023 4021 loistlesiAbilo ASSAiaiatat »2 ASabbObSbASblsbbAsiatat 22 Ab-BR-2051 87 4024 4022 oislesi asilsss aisisatat 22 SASlbbSbSbASbASbSASatat 22 Ab-OR-2052 96 84 76 69 87 4025 4023 csiloilbasslisib aatat 21 BSASABOBBSBASBASbblatat 22 Ab-OR-2053 92 84 79 76 74 40.37 4035 SABOBBALIBSSSB OSLBSATAT 93 SABSSAOOOAAbbsASASbbataG 24 Ab-TsR-2054 97 79 78 69 96 4038 4036 Aisiolliissssios 95 bbAboslobbAabbSlSabatat 22 Ab-TsR-2055 88 4039 4037 isislisssioslisAAbtit 22 ibOAbsblobsAAbbsACAAatat 22 Ab-PR-2056 sixteen 4040 4038 sisAAiississAisAAiotat 99 AibSabbSASobAabbSASOTaT w Ab-br-2115 4043 4041 laiisssssslismlilssatat 101 OsilibbAboslsbbAAbbatat 122 Ab-OR-2057 94 91 86 79 69 4051 4049 OSAISLI ASSA 103 blobbsibbbabiolbbsatat 125 Lb-RR-2058 86 4052 4050 sislils 105 sbAIAAbsisobAbbbAbbagat 106 Lb-br-2059 91 4057 4055 AilssAsoiylbylAlSalatate 107 AisiisbA Illssissilbatat 128 LY1R-2060 92 4058 4056 ilssAbsiiliAbAASAlsatat 102 obiobbsbAiAlosbbbbAatat 112 Ab-OR-2061 88 4059 4057 AsslussiaAilslASAASlatat 111 bbbbbibSbABaAbSbbbbatat 112 AYZR-2062 95 79 78 72 94 4060 4058 sslbsilyllslAASAAatot OF bbbiibbisbabl Absbbbatat 114 Ab-SR-2063 90 4061 4059 slosii or AOLASAA Allatate 115 biisibObbSbAbLabsbbatat 112 Ab OR-2064 94 86 76 67 83 4067 4065 LilolASLLSAAAAIIAislate 112 bbAbLabbiboibioiosabatag 112 Ab-PR-2065 91 4112 4110 orilASABALAAoyliobagate 112 ssAbAsiiiibsisibaAbAatat 122 Ab-TsR-2130 4251 4249 ib to Ailslibis; AOSAllat a g 121 biislisAAAbSbAbSbSAvtat 122 Ab-BR-2066 86 4252 4250 BASAilsliioolbOAAAsatat 1a SbbSabSaAAbbbAbSbSatat 124 Lb-br-2067 92 4254 4252 olilsliibiaiAiAlAssiase a g 125 AbObisAbslaAbObAbsatat 125 Ab-BR-2068 93 4255 4253 LiASAiiioloslAASsisatat 122 sloisiiisaisaaaobloiat 122 Ab-RR-2069 89 4256 4254 AndAsliis AibAalssossatag w SbAObbbslbsbaSAAAboyAatat 132 Α6ΌΡ-2074 4313 4311 AlbOblilsAALAASlbsAaatat 131 bbsiobbibibbAbOAsiiatat 132 Α6ΌΡ-2131 4314 4L2 loislilslaaaaslaslia ι at 133 AbOSbSbibibbiAbSASbatat 124 Α6ΌΡ-ΖΙ32 4316 4314 baublsaalalsabsababayt 135 AbAbbsBsiiibbObAbsAatat 132 Ab-PR-2133 4473 4471 IIIAabiAsilliaAbsiatat 122 AbSbADabbabbasibaalatat 132 Ab-TsR-2075 4474 4472 iilAb (1AsilAiiiAbsibatat 139 SAOSbAAAbbAObASbbAAatat 142 Λ6ΌΡ-2076 4475 4473 ilAbiAsilliiAbsiobatgg 141 sslasbALABbAbbASbblatat one" Ab-OT-2077 4476 4474 AAAAIASAIIilosiosAatat 143 BSSAbsilAabbAbilsibatat 144 Ab-BR-2078 4477 4475 lbiAsilAiinaiSIYOASiTat 145 bbssAsgblAabbloiASbatat 146 Ab-OR-2079 4478 4476 oiAsilAiiiAOosibbASA <pg <p 147 bbisSabsilAabbAbbASatat 142 Ab-br-2080 4480 4478 ASOAAiiilbsoooASAiiatat 149 AAbbissAbSbAAAiiAbstat 152 Α6ΌΡ-2081 44 $ Ι 4481 Aliyilbsioo ds 151 IssAAssissAOSblAAbiatat 152 Ab-BR-2082 4484 4482 liiAbsibbASliibbAOItat 153 AbslAbbisSSASSblAbatat 154 Lb-TsR-2083 4486 4484 ilbsisbls anibbaiisatitis 125 OAAissAAbbissAbsiAAatat 156 Ab-OR-2084 4519 4537 IiiioAAAAloAiiaoooolatat W bssssAAisiibisAAAAatat 152 Ab-OR-2134 4540 4538 iisistlAAAOAiiobSbAoata g 152 sissssAAbsibbbisAlaatat 160 Ab-OR-2135 4542 4540 IblAAAASAiiioosoAbAbatat one" SISBSSSSAAbsiiii Islat 122 ABOR-2136 4543 4541 olAAAAOliisboooAbAbbatat 163 sisbssssAlbSbbiiisatat 125 Ab-OR-2137 4671 4669 IbllSaciislbAisiiatat 1 "5 AbAbsibAASbSbiSabAatat 166 Ab-BR-2085 4672 4670 Aiollsiisloo Aisiisatat 122 baaoabsioalsbobbsbatsabatat 168 Λ6ΌΡ-2086 4867 4865 IssssiooboibiiAASlatat w bSbbAASALSSCAAOOSOSAOTOT 122 A1.-OG-2087 4868 4866 sssiiosoyoioyolASAiatat .171 AbSbbAASLASSSLAOOSbSbTaT 122 Ab-OR-2088 5544 5542 CAiAbsAiisliAObosAlazat w bbSASSbAbbAAbbSbAbAatat 174 AB PR-2089 5545 5543 AyAosAiisliAboiOAAOa gat 125 SbbSSASSbabbAaBosbabatat 126 Ab-PR-2090 5546 5544 IlosAiisAilsbibAboa gat 122 ssib sassb dibalbosilate g 178 Ab-PR-2091 5550 5548 AyisAilobiolAbbAbslatat 179 bosbssbbssassbbabaabat 122 Α6ΌΡ-2092

- 20 012573

5642

564)

5651

5640

5641

5638

5639

5640

5643

5644

5645

5647

5648

5649

5650

5642

5643

5645

5646

5647

5648

5752

5754

5755

5756

5919

5920 5934 60! 6

6019

6020

6252

6253

6254 iiusAlisAUsliAsiiildtat isslAtssAisliAoiiiAaggat sslliolisAUAOuiUASsatat <AAislisATsAsiiiASsi <gg (1t Alis.lisliAytsiilssilyatat AislisAilsiiiASsiliatdt SAUSAiliiirlssiliiodtdt AisATsAoiiiAssiliialdtdt uslilsiiUASsiAUUSAOstot sdils iiilssiliislsidtat AiloiiilssiliisAsiiitdt sliio ~ sosiiliiilslil <ya'dt iisoisuiAitstsASATsAiAdtit ISS; isiiAiiiASAiATsAA <pat SICC iliiilsAUAUAAldtd g lilisAiosislASAiolietdt NAislissisAAUATsOdiAdtat isliAitsoAiiisAAAiilitat ilsiilsussiiASAliAodtdt iiAsissiiASAAilosiSbGbt iAsissitsASAATsASsissdtit A IShkiAilssissASUibTbt CAIisclilosisoAsoildtdt AuisililossisASoyllottdt

W 1A W W

191 w 195

192

199 adG

2Y5 207 And

2Ts 213

212

217

219 cg

222

222

222 ilAAsiAisAisAiissAAdtat oiAAAsiAUSAisAtsissAetdt soiAAAsiA "oAisAiiosdt <gg ASsiAAASiAiolisAiioitdt tsAbsilAAsiAUSlisAtitat AUlssilAASUAUSAUSAUatat SAAUASSUALASiAOSAOSLdT islATsASoiAAASUAUSAiatat SiSAAUAOOUAAASUAUSADTDT AsisAAUASsilAlsiAUsatat AASUSAAUlosilAASUAUatat UAUsidAliAASAssAAisatat ULiAiSUAAAUAASASSAAaTDT UUAiAUaiAAAUAASASSlDTDT uuiATsAioiAAAUAASASsatdt disAisiisASSAisAUAiatat UAiSAUSUUOAOSAUSAiATsGdT UAAUUUilLAUSAAUAUSADTDT siAuisUAASSAsiAAOUAa'pl · UASsiAiiuiAAstsASillsTot SSASSUAiiSUAASSASUADTDT ASois ASsiAUASAATsAUbtat UASsissAdsiATsAOAASldtat iiASSUSSASSUAUASlAShTbt as * and?

AL-PP-2093

AL-PP-2094

AE-PP-2095

LRR-2096 ALR-2097 LE-RR-2098

AB-2138

AERR-2139

AE-PP-2140

AB-2099

AE-PP2100

AE-PP-2101

AE-PP-2102 AE-PP-2103 AE-OR-2141

AL-OR-2142

A1-PP2104 AE-PP-2105

AB-RR-2106

AB RR-210?

AE-OR-21S8

LRR-2109

A1-RG-2110

AB-2111

Table 1b

P gene Κ8Υ

Fact 5EO 10 ZE9 GO % inhibitory % inhibition % inhibition % inhibition % inhibition cheskoe Start ΝΟ-. N0: rations K5UA2 K5UA2 K51 / A2 ₽5УВ Start dot Semantic Antisense AG-OR # (5 nM) 500 pM 50 pM 5pM (5 nM) 55 53 AAdiissiAbAdisAdiAdotsL · 229 IIAiieAiishiABEAAiii <ggbt 230 A1-PP-2000 3 56 54 AAiissiASAAUSAAAAAbtat 231 iwiiAiiAaiisuAesAdiidtat 232 AB-2001 4 58 56 yissiASAAisAAiAAASbytt 233 ssiiiAiisAiishiAS <ZAA <yaat 224 AE-PP-2002 7 59 57 Issyasadisdai and adabosbot 235 Russia, Azerbaijan and Azerbaijan 236 AU-PP-2003 98 93 92 84 97 61 59 CuASAAiCAAiAAA6C6CA <1TaT 237 UssssiiiAiuoAiishiAootbt 238 A1-OR-2004 3 322 320 ASAUiibAiAASAAivAAbDTbT 239 siSaiUsiUAisAAAioibtat 240 AS-OR-2005 7 323 321 SAIUIEUUAAAAUSAASAOTOT 241 UsiusAiiviiAisAAAioshdt 2 " AB-2006 5 324 322 AiiioAiAASAAieAAbAAatat 243 jesius aiibia aisaaaidtcht 244 ΑΙ.-ΡΡ-2007 4 325 323 IIISAIAASAAIVAAAWAOTsgot 245 sisisiaiisiiaiisaaadtat 246 A1.-OR-2008 7 426 424 ABISAAAAIASUAbsAAiSdTdT SAiissiAOUAiiisAsiiatot 245 AB-2009 2 427 425 AoisAAAUASiAaedAiasspchp · 249 BSAIussiAeiAiiisAsibTbT 250 AS-PP-2010 7 428 "26 IibAAAAUASILSSALUSA.ITOT 251 ABSAiissiAeiAiiisASdott 252 AIRP-2011 4 429 427 IsAAAiAsiAbbAAyssiiat 253 AABSAiissiASUAIIisAatat 254 AG RR 2012 96 77 68 66 92 430 428 BAAAUASIASOAAibsiysdtdt 255 VAAasAiissiAsiAiiisatsp · 256 AG-RR-2013 98 85 76 75 89 431 429 AAAiAsiAsaAAUesuisAshchat 257 IsAAesAUissiAbuAiiuatdt 258 A1-PP-2014 98 85 81 68 66 550 548 SAASSAIIDDIS ASSAISPUT 2 §9 SAIISZBISAIIAAiosiosstat 260 A1-PP-2015 7 551 549 AASSAIIADISASSAAUSAYTOT 2 §1 ISAIIVSISAIIAAiosiosatot 262 AB-2016 98 88 82 75 94 SSAUAAiAiAASASSAASADTzDG 263 IsiuBsibiiAUAIIAisvatsdt 264 AG-OR-1729 90 Sat.AiidiAIIASAseAivAs1T5 <l 265 IsAissioiAAUAiAAUSbatsdg 266 AG-RR-1730

Table 1c

Ν gene Κ8Υ

_. OD YIGMYAM. % inhibition K5UA2 50 pM % inhibition of K5UA2 5lM % inhibition of κενβ (5nM) Actual start Semantic 8EO Yu BUT: Antisense 263 W AG-OR # % inhibitory level (5 nM) K8UA2 500 pM 3 sectionsAbsLAAsisAAB <jas1t 267 SyisAsiiiSidSASASSSbtsp · 268 AG-OR-2017 98 86 84 80 93 5 Sisi idea aa AsisAAaviibtat 269 AASIIASIIissidAvAbatdt 270 AE-OP-2018 2 52 sieisAissASSAAAidsAbTegg 271 υουΑυυυοουοβΑυοΑΟΑβάΤάτ 272 AB-2019 5 $ 3 IsisdissdsdsdsAidsASotat 273 sushiAiiissisbAizdSAOotat 274 AE-OP-2020 2 191 iAdiAbsiieiUAiAissbTdt 275 OsAiAUAASAiAssiAiidsggdt 276 AE-OR-2021 3 379 AiibASDiA6AAUSiASAAs1Ts1T 277 iisiASAiisiAisisdiatat 278 AE-OR-2022 98 78 77 75 94 897 AiisilssdidiAiisAASutsp · 279 SiisAAiAiAussiAbAAiatat 280 AG-OR-2023 one 898 IsidssAiAiAiiioAASAbtst 281 IsisAAiAidissiAOAAatsp · 262 AG-OR-2024 7 899 ISUACCAidiAiisAACAA <1Tc1T 283 UisiAAAUASAissiAsa ^ tat 284 AE-OR-2025 96 89 84 77 96

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table 2

Table 3

one A1YUR2017 10 nM Protein% 0 Activity% one hundred 2 10 nM 0 one hundred 3 100 pM 0 one hundred 4 10 pM eleven,. 7 8 88.22 5 1 pM 70.63 29.37 6 100 fM 72.7 27.3 7 The control PB5 one hundred 0 8 2153 10 nM 94.54 4,5

CLAIM

Claims (4)

CLAIM 1. A method of reducing the levels of protein of respiratory syncytial virus (CJA), K8U mRNA or K8U titer in the patient’s lung or respiratory cells, comprising the step of intranasal administration or direct injection of 81RNA agent into the patient’s lungs, where the 81RNA agent contains a sense strand consisting of a sequence 8EO GO N0: 267, and an antisense chain consisting of the sequence 8EP GO N0: 268. 2. The method according to claim 1, where the specified agent 81RNA agent is administered to the patient by inhalation or nebulization. 3. The method according to claim 1, where the specified 81RNA agent reduces the titer of the virus in the specified patient. 4. The method according to claim 1, where the patient is diagnosed with a viral infection caused by K8U.